Ergot alkaloids improve human health as powerful and versatile pharmaceuticals for treatment of multiple conditions including senile dementia, Alzheimer's disease, Parkinson's disease, migraines, hyperprolactinemia, and type 2 diabetes. Different types of ergot alkaloids are produced by distantly related groups of fungi. Certain species of Claviceps and Epichlo produce ergot alkaloids derived from lysergic acid, and these have powerful pharmacological activity. Two other species of Claviceps make dihydroergot alkaloids, in which slightly different chemistry results in different but equally powerful activitis. A third group of fungi, including Aspergillus fumigatus, makes a family of ergot alkaloids that is no derived from lysergic acid or dihydrolysergic acid and is not used clinically; however, A. fumigatus grows faster, is more readily manipulated, and has greater potential for genetically improving ergot alkaloids. All these fungi share common early steps in their ergot alkaloid pathways before diverging to make their unique end products. By blocking the A. fumigatus pathway at a specific point and adding two selected genes from a lysergic acid producer, A. fumigatus has been modified to produce lysergic acid. The long-term goals of the proposed project are to modify and expand the A. fumigatus expression system to answer basic questions about the origins of lysergic acid and related compounds. In addition to answering basic genetic questions, the work will have practical healthcare-related implications, because modified fungal strains will produce important ergot alkaloids that are rare in nature but are pharmaceutically relevant compounds. Specific aims are to (1) Clarify steps in the production of lysergic acid and elucidate the biosynthetic origins of alternate ergot alkaloids paspalic acid and lysergol, and (2) Determine the biosynthetic origin of dihydroergot alkaloids. Individual genes or combinations of two genes will be isolated from fungi that make these unique ergot alkaloids and expressed in A. fumigatus modified to produce one of two different substrate ergot alkaloids. The A. fumigatus strains transformed with these genes will be analyzed by molecular and biochemical methods to determine how expression of the candidate genes has affected the fungus's ergot alkaloid profile. Results of the proposed project will reveal roles of specific genes and elucidate origins of several important ergot alkaloids. The project will produce strains of A. fumigatus that produce molecules with pharmaceutical significance. Additional benefits include meaningful experiences for graduate and undergraduate students and further development of a platform for modification and improvement of additional or novel ergot alkaloids.